A taxol ganoderma spore oil self-nanoemulsion composite microparticle with ganoderma spores as a carrier, and a preparation method and use thereof

Abstract

The present application relates to a kind of ganoderma lucidum spore as carrier paclitaxel ganoderma lucidum spore oil self-nanoemulsion composite microparticle and its preparation method, belong to medical technology field.The pretreated ganoderma lucidum spore (GLS) is obtained by process screening, and it is used as drug carrier, and the paclitaxel ganoderma lucidum spore oil self-nanoemulsion (PGS) is loaded therein, and paclitaxel composite microparticle (PGS@GLS) is obtained.Release curve in vitro shows that PGS@GLS has the characteristics of slow release after reaching intestinal tract;In vivo pharmacodynamics experiment shows that PGS@GLS can significantly inhibit the development and metastasis of colorectal cancer in mice;Tumor tissue section immunofluorescence analysis shows that PGS@GLS can effectively exert the therapeutic effect on colon cancer.Paclitaxel self-nanoemulsion ganoderma lucidum spore composite microparticle promotes cancer immune cycle by inducing immunogenic cell death and immune regulation, significantly improves the treatment effect of colon cancer, and has good biological safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a paclitaxel Ganoderma lucidum spore oil self-nanoemulsion composite microparticle with Ganoderma lucidum spores as a carrier and a preparation method thereof. BACKGROUND

[0002] Colorectal cancer (CRC) is a common digestive tract cancer originating from the epithelial tissue of the large intestine, mainly occurring in middle-aged and elderly people over 40 years old, and has a high mortality rate. Lymph node metastasis is one of the most important reasons for poor prognosis of patients with colorectal cancer. However, conventional chemotherapy or other oral drugs are difficult to reach the lymph nodes, and cannot effectively inhibit the occurrence and development of micro-metastases therein.

[0003] The occurrence of cancer is the result of gene mutation and change in normal cell regulation process. The body's own immunity will produce a response and a series of reactions, including: tumor cells release tumor antigens, which are captured by dendritic cells and presented to T cells, initiating and activating effector T cells to respond to cancer-specific antigens, activated effector T cells transport and infiltrate tumor lesions, specifically recognize and bind to cancer cells, and ultimately kill tumor cells; and the killed cancer cells release more tumor-associated antigens; the above process is repeated in a cycle, which is called cancer immune cycle. However, without drug intervention, the tumor site may fail to effectively initiate and operate the above process due to lack of antigens and presentation, ultimately leading to continuous development and metastasis of the tumor.

[0004] Paclitaxel (PTX) is a tetracyclic diterpenoid organic compound extracted and separated from natural Taxus chinensis bark, which plays a role in inhibiting tumor cell proliferation by disrupting the dynamic balance of continuous polymerization and depolymerization between tubulin and microtubules, and has good antitumor effect. It is clinically used in the form of injection for the treatment of various cancers. However, injection administration brings great inconvenience to patients and causes hypersensitivity reactions and serious side effects in many patients. However, the poor water solubility and permeability of paclitaxel make the development of its oral preparation face many problems. In addition, paclitaxel resistance is one of the important factors affecting its effective antitumor effect. Therefore, it is crucial to improve the drug efficacy by compounding other active ingredients and to effectively deliver it to the body by improving its solubility and permeability through formulation.

[0005] Ganoderma lucidum spore oil (GLSO) is a light yellow to yellow clear oil liquid extracted from the reproductive cells of Chinese medicine Ganoderma lucidum, i.e. Ganoderma lucidum spores. The main components include Ganoderma triterpenes and fatty acids. GLSO has the effects of inhibiting tumor cell proliferation and regulating immunity. The mechanism of its immune regulation is related to enhancing the phagocytic ability of macrophages, increasing the number of lymphocytes, improving the CD4+ / CD8+ T cell ratio, promoting the maturation of dendritic cells, and increasing the levels of IFN-γ, TNF-α, IL-1 and other cytokines in the body. However, GLSO is not easy to store and is prone to oxidation. Its effect is limited by its low water solubility. For some special groups of people, GLSO is also difficult to absorb due to insufficient bile secretion, thereby affecting its full immune regulation effect. Therefore, improving its solubility and promoting its effective absorption through preparation is the premise of fully exerting its therapeutic effect.

[0006] Ganoderma lucidum spore (GLS) is the reproductive cell of Chinese medicine Ganoderma lucidum, which has a double-walled structure of oval cysts with a diameter of about 5 μm. There is a rich β-glucan in the cell wall of Ganoderma lucidum spore, which cannot be digested in the stomach and small intestine and is degraded by bacterial fermentation in the colon. It has been reported that β-glucan is selectively recognized by receptors such as Dectin-1, complement receptor 3 (CR3) or Toll-like receptor (TLRs) expressed on the surface of immune cells as a pathogen-associated molecular pattern, specifically binds to immune cells, stimulates the occurrence of adaptive immune response, and then plays an anti-tumor role.

[0007] Design of composite microparticle drug delivery system loaded with multiple active ingredients, simultaneous delivery of chemotherapeutic drugs and immunomodulatory active components, is one of the effective strategies to promote cancer immune circulation and improve the therapeutic effect of drugs on tumors. Self-nanoemulsifying drug delivery system (SNEDDS) is a uniform liquid composed of oil phase, drug and surfactant, which can be rapidly self-emulsified into nanoemulsion upon contact with water, then transported across the epithelium through lipolysis, and finally excreted from the cell in the form of chylomicron, into the chylomicron and lymphatic system, thereby increasing the lymphatic transport of drugs and improving the oral bioavailability of poorly soluble drugs. The key feature of using SNEDDS to simultaneously deliver poorly soluble chemotherapeutic drug paclitaxel and immunomodulatory active component ganoderma spore oil is to use the active component as the oil phase required for the preparation of the formulation, and to improve the solubility of the two drug components and the delivery efficiency by combining drug and adjuvant. Then, the modified ganoderma spores are used as a natural drug reservoir, and the drug is loaded by vacuum adsorption to improve the stability of the drug and effectively avoid the premature emulsification of paclitaxel and ganoderma spore oil in the stomach, thereby increasing the drug absorption through the intestinal lymphatic system and improving the therapeutic effect on colorectal cancer.

[0008] The delivery of paclitaxel formulations and their therapeutic effect on tumors in the prior art still need to be further studied and improved. SUMMARY

[0009] The purpose of the present application is to provide a paclitaxel-loaded ganoderma spore composite microparticle for treating colorectal cancer and a preparation method thereof. The paclitaxel-loaded ganoderma spore composite microparticle prepared by the present application can simultaneously deliver chemotherapeutic drugs and immunomodulatory active components, improve the solubility of the drugs, and promote the absorption of the drugs through the oral lymphatic transport pathway.

[0010] The drugs and adjuvants involved include paclitaxel, ganoderma spore oil, emulsifiers and co-emulsifiers.

[0011] Among them, paclitaxel is a clinically recognized chemotherapeutic drug with the effect of causing immunogenic cell death of tumor cells; ganoderma spore oil and ganoderma spores have the effect of stimulating immune regulation; the emulsifier has the characteristics of gelling and emulsifying, which can promote the self-nanoemulsion to undergo a gel state before emulsification, combined with the porous loading of ganoderma spores, which can avoid the premature emulsification and release of the drug in the stomach, thereby more absorption through the intestinal lymphatic pathway into the body.

[0012] The application utilizes a self-emulsifying drug delivery system to prepare a paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion, effectively improves the solubility of the two drugs by screening suitable emulsifiers and co-emulsifiers, and obtains hollow porous but still intact blank Ganoderma lucidum spore microparticles through acid and alkali multi-step treatment, and then loads the drug-loaded self-nanoemulsion into the Ganoderma lucidum spore microparticles through negative pressure adsorption, thereby obtaining paclitaxel-loaded Ganoderma lucidum spore composite microparticles.

[0013] The application is achieved by the following technical solutions:

[0014] A paclitaxel-loaded Ganoderma lucidum spore composite microparticle is characterized by being composed of two parts: paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion (PGS) and Ganoderma lucidum spore (GLS).

[0015] The mass ratio of the Ganoderma lucidum spore and the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion is 1:5-10.

[0016] The paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion comprises the following components:

[0017] (a), paclitaxel (PTX);

[0018] (b), Ganoderma lucidum spore oil (GLSO);

[0019] (c), an emulsifier, which is selected from one of polyoxyethylene 40 hydrogenated castor oil (RH40), Tween 80, lauryl polyoxyethylene-32 glycerol ester or caprylic capric acid polyethylene glycol glycerol ester;

[0020] (d), a co-emulsifier, which is selected from one of monolauric acid propylene glycol ester, polyethylene glycol 400, 1,2-propylene glycol, diethylene glycol monoethyl ether or propylene glycol monolaurate.

[0021] In the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion, the PTX content is 0.08-0.64% by mass, the GLSO content is 10-40% by mass, the emulsifier content is 27-72% by mass, the co-emulsifier content is 14-60% by mass, the stirring speed is 200-600 rpm, and paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion (PTX-GLSO SMEDDS, PGS) is obtained.

[0022] The average emulsification particle size of PGS is about 26 nm, the polydispersity coefficient (PDI) is about 0.08, the drug loading (calculated based on paclitaxel) ranges from 0.15% to 1.41%, and the encapsulation rate can be as high as 90.53%.

[0023] The preparation method of the paclitaxel-loaded Ganoderma lucidum spore composite microparticle comprises the following steps:

[0024] (a), preparation of a paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion (PGS) pre-concentrate:

[0025] Weigh the prescription proportion of GLSO, emulsifier and co-emulsifier, and stir them at room temperature to make them completely mixed and uniform, to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate containing 10-40% of GLSO by mass content;

[0026] Add the prescription proportion of PTX to the above pre-concentrate, and stir in the dark to make it completely dissolved and mixed uniformly, to obtain PGS.

[0027] (b) Preparation of defatted Ganoderma lucidum spores:

[0028] The defatting method is selected from one of solvent extraction method, ultrasonic-assisted extraction method, supercritical CO2 extraction method, and gradient centrifugation method. Hereinafter, the solvent extraction method is taken as an example for detailed step description:

[0029] Disperse a certain mass of Ganoderma lucidum spores in a certain volume of acetone (control the proportion of acetone to spores to be 10-20:1 (V / W)), unit mL / g, stir and reflux for 8-24 h, control the stirring speed to be 200-600 rpm, and control the reflux temperature to be 58-65℃;

[0030] Remove the organic solvent in the above reflux mixture by suction filtration, and dry naturally.

[0031] (c) Alkaline-treated Ganoderma lucidum spore particles:

[0032] Disperse the defatted Ganoderma lucidum spores dried naturally in a potassium hydroxide solution of a certain concentration (control the proportion of dried spores to alkali solution to be 1:20-25 (W / V)), and the concentration of potassium hydroxide ranges from 6% to 18%;

[0033] Stir in a water bath for 2-6 h, the water bath temperature is 70-80℃, and the stirring speed is 200-600 rpm;

[0034] Centrifuge at 3500-4500 rpm for 15-20 min, discard the supernatant, and wash with hot distilled water for 3-6 times to remove the alkali-soluble impurities.

[0035] (d) Acid-treated Ganoderma lucidum spore particles:

[0036] Disperse the alkaline-treated Ganoderma lucidum spores in phosphoric acid (control the proportion of spores to phosphoric acid to be 1:15-1:25 (W / V)), and the mass concentration of phosphoric acid is 80-85%, stir in a water bath for 2-6 h, the water bath temperature is 40-50℃, and the stirring speed is 200-600 rpm.

[0037] (e) Water-washed Ganoderma lucidum spore particles:

[0038] The spore phosphoric acid suspension is dispersed in hot distilled water, the water temperature is controlled at 60-65°C, the ratio of the original spore mass to the volume of distilled water is controlled at 1:100-1:120, and stirring is performed until complete mixing is achieved;

[0039] Centrifugation is performed at 3500-4500 rpm for 15-20 min, the supernatant is discarded, and the above operation is repeated 3-6 times to remove acid-soluble impurities and residual phosphoric acid, until the pH of the supernatant is close to neutral (pH 6.0-7.0).

[0040] (f) alcohol-washed Ganoderma spore microparticles:

[0041] The water-washed Ganoderma spores are dispersed in anhydrous ethanol (the ratio of the spores to anhydrous ethanol is controlled at 1:10-1:20 (W / V)), and stirring is performed until complete dispersion is achieved.

[0042] Centrifugation is performed at 3500-4500 rpm for 15-20 min, the supernatant is discarded, and the above operation is repeated 2-4 times.

[0043] (g) dried Ganoderma spore microparticles:

[0044] The alcohol-washed spores are dried in a reduced-pressure drying oven under reduced pressure until a constant weight is achieved (the temperature is controlled at 60-65°C), and the Ganoderma spore blank carrier is obtained.

[0045] (h) drug loading: vacuum negative pressure drug loading:

[0046] The drug loading method is selected from one of vacuum reduced pressure method, stirring method and centrifugation method;

[0047] Preferably, the drug loading method is selected as the vacuum reduced pressure method, which is simple and easy to operate and is suitable for large-scale preparation. The following is a detailed step-by-step description of the vacuum reduced pressure method.

[0048] The paclitaxel Ganoderma spore oil self-nanoemulsion pre-concentrate and the blank Ganoderma spore microparticles are vortex-mixed, and the mass ratio of the two is controlled at 5-10:1.

[0049] The mixture is placed in a vacuum drying oven, the temperature is controlled at 28-32°C, the pressure is controlled at -0.01-0.08 MPa, and the mixture is maintained for 2-4 h.

[0050] The vacuum-loaded self-nanoemulsion and spore mixture is taken out, centrifuged at 3500-4500 rpm for 15-20 min, and the lower layer of drug-loaded spores is collected. The surface of the spores is rinsed with ethanol, and the spores are naturally air-dried at room temperature to obtain the paclitaxel Ganoderma spore oil self-nanoemulsion Ganoderma spore composite microparticles (PGS@GLS).

[0051] The obtained PGS@GLS needs to be stored in a sealed environment at ≤25°C.

[0052] Compared with the prior art, the present application has the beneficial effects that:

[0053] (1) The technical obstacle for loading the liquid self-nanoemulsion into the porous hollow ganoderma spore sac is the leakage problem of the self-nanoemulsion. The self-nanoemulsion prepared in the present application uses a specific emulsification system, and after entering the body with the ganoderma spore, it will be in a gel state after encountering the water environment, effectively preventing the premature release of the drug from the spore.

[0054] (2) In terms of drug efficacy, the combination of paclitaxel, ganoderma spore oil and ganoderma spore can significantly improve the expression of calreticulin in tumor tissue, the infiltration of mature dendritic cells, and the proportion of CD8+ T lymphocytes and mature dendritic cells in mesenteric lymph nodes. The above-mentioned calreticulin is a marker protein of immunogenic cell death (the first step of cancer immune cycle), mature dendritic cells play an antigen presenting role (the second step of cancer immune cycle), CD8+ T lymphocytes are important immune cells for eliminating tumor cells (the third and fourth steps of cancer immune cycle), which work together to promote immunity and enhance anti-tumor effect.

[0055] (3) Based on the oral absorption characteristics of self-nanoemulsion, by loading the paclitaxel ganoderma spore oil self-nanoemulsion into the ganoderma spore microcapsule, the drug can be released more slowly and persistently, and the concentration of the drug in the mesenteric lymph nodes is increased, thereby improving the treatment effect of the drug on intestinal tumors. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 A dried ganoderma spore before treatment (left) and after treatment (right);

[0057] Figure 2 A is the morphology of ganoderma spore before treatment under inverted phase contrast microscope, Figure 2 B is the morphology of ganoderma spore after treatment under inverted phase contrast microscope;

[0058] Figure 3 A is the scanning electron microscope image of ganoderma spore before treatment, Figure 3 B is the scanning electron microscope image of ganoderma spore after treatment;

[0059] Figure 4 A is the transmission electron microscope image of ganoderma spore before treatment, Figure 4 B is the transmission electron microscope image of ganoderma spore after treatment;

[0060] Figure 5 It is the appearance of self-nanoemulsion taken by transmission electron microscope;

[0061] Figure 6 It is the in vitro cumulative release diagram of paclitaxel in PGS@GLS and PTX@GLS (mean ± SD, n = 6);

[0062] Figure 7 A is a schematic diagram of the change of drug fluorescence distribution in the mesenteric lymph nodes in vivo over time, Figure 7 B is a graph of the average fluorescence intensity in the mesenteric lymph nodes in vivo over time;

[0063] Figure 8 A is a representative picture of the results of in vivo imaging of animals in each group (n = 6), Figure 8 B is a graph of the average fluorescence intensity in the abdomen of mice in the model group over time, Figure 8 C is a graph of the average fluorescence intensity in the abdomen of mice in the PTX i.v. group over time, Figure 8 D is a graph of the average fluorescence intensity in the abdomen of mice in the GLS group over time, Figure 8 E is a graph of the average fluorescence intensity in the abdomen of mice in the PGS group over time, Figure 8 F is a graph of the average fluorescence intensity in the abdomen of mice in the PGS@GLS group over time;

[0064] Figure 9 is the change in body weight of mice;

[0065] Figure 10 is the score result of the physical condition of mice (mean ± SD, n = 6);

[0066] Figure 11 A is an anatomical diagram of the development of intestinal tumors in mice in each administration group, Figure 11 B is a statistical graph of the number of intestinal tumors in mice in each administration group, Figure 11 C is an analysis of the mass of tumors in the colorectal region of mice in each administration group, mean ± SD, n = 3);

[0067] Figure 12 A is a schematic diagram of the flow cytometry analysis results of mature dendritic cells in the tumor tissue of mice in each administration group; Figure 12 B is a schematic diagram of the flow cytometry analysis results of CD8+ T cells in the tumor tissue of mice in each administration group; Figure 12 C is the quantitative results of mature dendritic cells in the tumor tissue of mice in each administration group; Figure 12 D is the quantitative results of mature dendritic cells in the mesenteric lymph nodes of mice in each administration group; Figure 12 E is the quantitative results of CD8+ T cells in the tumor tissue of mice in each administration group; Figure 12 F is the quantitative results of CD8+ T cells in the mesenteric lymph nodes of mice in each administration group.

[0068] Figure 13 is the immunofluorescence staining results of calreticulin and HMGB1 in the tumor tissue of mice in each administration group (scale bar = 50 μm);

[0069] Figure 14The immunofluorescence staining results of cell proliferation and apoptosis in tumor tissues of each administration group (scale bar = 50 μm);

[0070] Figure 15 The CD8+ T lymphocyte infiltration in tumor tissues of each administration group;

[0071] Figure 16 The white blood cell (WBC), lymphocyte (Lymph), platelet (PLT), red blood cell (RBC), and hemoglobin (HGB) count statistics of mice in each administration group (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0072] Figure 17 The H&E staining results of the main organs and tumor tissues of mice in each administration group;

[0073] Figure 18 Schematic diagram of PGS@GLS composite microparticles activating cancer immune circulation and inhibiting the development of colorectal cancer. DETAILED DESCRIPTION

[0074] Example 1

[0075] 1) Precisely weigh 20 g of Ganoderma lucidum spores, place them in 400 mL of acetone, and stir and reflux at 65°C for 12 h. Filter and air dry to obtain the defatted Ganoderma lucidum spores;

[0076] 2) Weigh 16 g of the dried Ganoderma lucidum spores after acetone defatting, and add them to 400 mL of a 12% (W / V) potassium hydroxide solution. Stir and reflux at 80°C in a water bath for 2 h. Centrifuge at 4200 rpm for 15 min, discard the supernatant, and resuspend in 60°C distilled water. Stir and wash, centrifuge, and repeat three times to obtain the alkali-treated Ganoderma lucidum spores;

[0077] 3) Redisperse in 320 mL of phosphoric acid, and stir at 40°C in a water bath for 2 h to obtain the alkali- and acid-treated Ganoderma lucidum spores;

[0078] 4) Disperse the above-mentioned spore phosphoric acid suspension in 2000 mL of hot distilled water (60°C), and stir for 0.5 h until completely mixed and uniform. Centrifuge at 4200 rpm for 15 min, discard the supernatant, and repeat the water washing operation three times. The pH of the supernatant is near neutral;

[0079] 5) Redisperse the above-mentioned water-washed Ganoderma lucidum spores in 200 mL of anhydrous ethanol, stir until dispersed uniformly, centrifuge at 4200 rpm for 15 min, and discard the supernatant. Repeat the above-mentioned operation two times;

[0080] 6) The spore precipitate was dispersed in 50 mL of absolute ethanol, and volatilized at room temperature until no obvious liquid remained. The treated spores were then transferred to a vacuum drying oven and dried at 60°C under reduced pressure until a constant weight was obtained. The treated Ganoderma spores were loose and dispersed, and the color was slightly darker. The results are shown in Table 1. Figure 1 .

[0081] The GLS of Example 1 was observed under an inverted microscope. Compared with untreated Ganoderma spores, the GLS was hollow, the double-layer wall was thinner, and one end of the spore was open. The results are shown in Table 2. Figure 2 .

[0082] Example 2

[0083] Precisely 5150 mg of RH40, 3000 mg of L90, and 1850 mg of Ganoderma spore oil were weighed, and magnetically stirred at room temperature until completely mixed and uniform. A self-nanoemulsion pre-concentrate of Ganoderma spore oil was obtained.

[0084] Example 3

[0085] Precisely 14.8 mg of paclitaxel was weighed and added to the self-nanoemulsion pre-concentrate of Ganoderma spore oil obtained in Example 2. The mixture was magnetically stirred at room temperature in the dark until completely dissolved, and a paclitaxel-Ganoderma spore oil self-nanoemulsion pre-concentrate (PGS) was obtained.

[0086] Example 4

[0087] 1) Precisely 7.4 mg of paclitaxel was weighed and dissolved in 1000 μL of absolute ethanol, and then mixed with 1000 mg of GLS from Example 1. The mixture was vortexed until completely mixed and uniform.

[0088] 2) The mixture was placed in a vacuum drying oven and maintained at -0.01 MPa for 2 h.

[0089] 3) After removal, the mixture was centrifuged at 4200 rpm for 15 min, and the lower layer of drug-loaded spores was collected. The drug-loaded spores were washed with 95% ethanol and finally air-dried at room temperature to obtain paclitaxel-loaded Ganoderma spores (PTX@GLS).

[0090] Example 5

[0091] 1) 5000 mg of the self-nanoemulsion pre-concentrate of Ganoderma spore oil from Example 2 was mixed with 1000 mg of GLS from Example 1, and vortexed until completely mixed and uniform.

[0092] 2) The mixture was placed in a vacuum drying oven and maintained at -0.01 MPa for 2 h.

[0093] 3) After removal, the mixture was centrifuged at 4200 rpm for 15 min, the drug-loaded spores in the lower layer were collected, washed with 95% ethanol, and finally dried naturally at room temperature to obtain Ganoderma lucidum spore composite microparticles loaded with Ganoderma lucidum spore oil nanoemulsion (GS@GLS).

[0094] Example 6

[0095] 1) 5000 mg of the paclitaxel Ganoderma lucidum spore oil nanoemulsion preconcentrate prepared in Example 3 was taken and mixed with 1000 mg of the GLS prepared in Example 1, and vortexed until completely mixed;

[0096] 2) Place the above mixture in a vacuum drying oven at -0.01 MPa for 2 h;

[0097] 3) After removal, centrifugation was performed at 4200 rpm for 15 min, and the drug-loaded spores in the lower layer were collected, washed with 95% ethanol, and finally dried naturally at room temperature to obtain paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion Ganoderma lucidum spore composite particles (PGS@GLS).

[0098] Example 7

[0099] 4500 mg of Tween 80, 3000 mg of L90, and 2500 mg of Ganoderma lucidum spore oil were accurately weighed and magnetically stirred at room temperature until completely mixed to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0100] 20 mg of paclitaxel was accurately weighed and placed in the above-prepared Ganoderma lucidum spore oil self-nanoemulsion preconcentrate, and the mixture was stirred under magnetic stirring at room temperature in the dark until it was completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion preconcentrate (PGS).

[0101] The method for preparing paclitaxel-Ganoderma lucidum spore oil-loaded Ganoderma lucidum spore composite microparticles from paclitaxel-Ganoderma lucidum spore oil-self-nanoemulsion pre-concentrate is the same as that in Example 6.

[0102] Example 8

[0103] 5250 mg of lauroyl polyoxyethylene 32-glyceride, 3000 mg of L90, and 1750 mg of Ganoderma lucidum spore oil were accurately weighed and magnetically stirred at room temperature until completely mixed to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0104] 28 mg of paclitaxel was accurately weighed and placed in the above-prepared Ganoderma lucidum spore oil self-nanoemulsion preconcentrate, and the mixture was stirred under magnetic stirring at room temperature in the dark until it was completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion preconcentrate (PGS).

[0105] The method for preparing paclitaxel-Ganoderma lucidum spore oil-loaded Ganoderma lucidum spore composite microparticles from paclitaxel-Ganoderma lucidum spore oil-self-nanoemulsion pre-concentrate is the same as that in Example 6.

[0106] Example 9

[0107] Precisely weigh 4700 mg of caprylic capric acid polyethylene glycol glyceride, 2300 mg of Span 80, 3000 mg of Ganoderma lucidum spore oil, and magnetically stir at room temperature until completely mixed and uniform to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0108] Precisely weigh 48 mg of paclitaxel, and place it in the Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate obtained above, and magnetically stir at room temperature in the dark until completely dissolved to obtain a paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate (PGS).

[0109] The method for preparing paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion loaded Ganoderma lucidum spore composite microparticles from the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate is the same as in Example 6.

[0110] Example 10

[0111] Precisely weigh 6430 mg of RH40, 2144 mg of PEG400, and 1429 mg of Ganoderma lucidum spore oil, and magnetically stir at room temperature until completely mixed and uniform to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0112] Precisely weigh 30 mg of paclitaxel, and place it in the Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate obtained above, and magnetically stir at room temperature in the dark until completely dissolved to obtain a paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate (PGS).

[0113] The method for preparing paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion loaded Ganoderma lucidum spore composite microparticles from the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate is the same as in Example 6.

[0114] Experimental Example 1

[0115] The untreated Ganoderma lucidum spores and the acid-base treated Ganoderma lucidum spores (GLS) samples were respectively dispersed uniformly with 95% ethanol, adhered to conductive glue, and gold sprayed, and the morphology of the Ganoderma lucidum spores before and after treatment was observed and compared by scanning electron microscopy. It can be observed that the untreated Ganoderma lucidum spores are oval-shaped with a size of about 5x8 μm, have a navel at one end, and have a smooth surface with slight closed-mouth depression. After acid-base water washing treatment, the spore size is slightly reduced to about 4x7 μm, the surface is rough, has a porous structure, and the navel end of the spore is open and broken. The results are shown in Figure 3 .

[0116] Experimental Example 2

[0117] A small amount of untreated Ganoderma spores and PGS@GLS obtained in Example 6 were dispersed uniformly with 95% ethanol, 10 μL of each was dropped onto a copper mesh and dried; transmission electron microscopy was used to take transmission images at 80 kV voltage to observe the morphology of the drug-loaded Ganoderma spore composite microparticles; it can be directly observed that the untreated Ganoderma spores are opaque due to the complete spore biological structure (including polysaccharides, proteins and other substances) inside; the transparency of the pictures of the Ganoderma spores treated with acid and alkali increased significantly, the outline shape was basically unchanged but there were multiple transparent holes in the middle, indicating that the internal substances of the spores can be effectively removed during the treatment process such as acid and alkali washing, the spore wall forms small holes, but the basic structure of the spore shell is not damaged, and the results are shown in Figure 4 .

[0118] Experimental Example 3

[0119] The sample obtained in Example 3 was dispersed in deionized water, and the average particle size, PDI, drug loading and encapsulation efficiency were determined, and the results showed that the average particle size of the paclitaxel Ganoderma spore oil self-nanoemulsion prepared was (26.160 ± 0.734) nm, the PDI was 0.022 ± 0.013, and the drug loading and encapsulation efficiency (calculated as paclitaxel) were 1.41% and 90.53%, respectively.

[0120] Experimental Example 4

[0121] The emulsified sample obtained in Experimental Example 3 was dropped onto a transmission electron microscopy copper mesh, negatively stained with phosphotungstic acid, and dried before imaging with a transmission electron microscope, and the results showed that the transmission electron microscopy images of the emulsion droplets after self-nanoemulsion emulsification showed uniform circular imprints, and the size was consistent with the results determined by the nanoparticle size analyzer, and the results are shown in Figure 5 .

[0122] Experimental Example 5

[0123] The drug-loaded spores in Example 4 and Example 6 were sequentially immersed in buffer solutions with pH 1.2, pH 6.8 and pH 7.4, the temperature was set to 37°C, and samples were taken at 2h, 3h, 5h, 7h, 8h, 12h and 24h, and the cumulative release amount (%) of paclitaxel at different time points was determined, and the results showed that the cumulative release amount of paclitaxel in Example 4 was less than 20% in the first 2h (pH 1.2), and then it was released slowly, the cumulative release amount was more than 80% at 8h (pH 7.4), and more than 90% at 12h (pH 7.4), and the cumulative release amount of the drug-loaded spores in Example 6 was less than 50% at 12h, and the results are shown in Figure 6 , which indicates that after the paclitaxel Ganoderma spore oil self-nanoemulsion is loaded into the Ganoderma spore microcapsules, the drug can be slowly released, and more paclitaxel in a dissolved state can be detected in the release medium after the paclitaxel is prepared as a self-nanoemulsion, indicating that the dissolution capacity is effectively improved.

[0124] Experimental Example 6

[0125] 1) Take 54 healthy male 6-8 week old Kunming mice, with an average weight of about 18-22g, adaptively feed for one week, and randomly divide the animals into 3 groups before the experiment, 18 in each group, weigh and mark;

[0126] 2) The 3 groups of mice were respectively given free DiR (Free DiR), DiR-labeled paclitaxel Ganoderma spore oil self-nanoemulsion (PGS) group and DiR-labeled paclitaxel Ganoderma spore composite microparticle (PGS@GLS) by gavage;

[0127] 3) Collect the mesenteric lymph nodes of each group of mice at 1, 2, 4, 6, 12, 24h after administration, and detect the fluorescence intensity of the mesenteric lymph nodes of different groups of mice at different time points by small animal imaging system. The results are shown in Figure 7 , The fluorescence intensity of the mesenteric lymph nodes of the PGS group and the PGS@GLS group mice at each time point was significantly stronger than that of the free DiR group, among which the fluorescence intensity of the PGS group was the highest at 2h, and the fluorescence intensity of the PGS@GLS group reached the peak at 6h, and remained at a high intensity at 24h after administration. The increase of fluorescence intensity indicates that the self-nanoemulsion preparation is beneficial to the absorption of more drugs into the mesenteric lymph nodes; the difference in the peak change time of fluorescence intensity reflects the difference in drug release and absorption in vivo, and the peak of fluorescence distribution in the lymph nodes after oral administration of the PGS@GLS group is later than that of the PGS group, indicating that the self-nanoemulsion after being loaded with GLS will be released more slowly and persistently in vivo.

[0128] Experimental Example 7

[0129] 1) Take 40 healthy male 6-8 week old Balb / c mice, with an average weight of about 18-22g, adaptively feed for one week, and then weigh and mark the animals after fasting for 12h without water the day before modeling;

[0130] 2) After the animals were anesthetized, they were fixed in a supine position, the skin was prepared and disinfected, a small incision was made on the abdomen and the cecum was exposed, and 2x10 7 luc-CT26 cells were injected into the cecum wall using a 30G needle, and then sutured;

[0131] 3) The mice were randomly divided into 5 groups, including the model group (C), the commercially available paclitaxel injection group (PTX), the blank ganoderma spore (GLS) group, the paclitaxel ganoderma spore oil self-nanoemulsion (PGS) group and the paclitaxel-loaded ganoderma spore composite microparticle (PGS@GLS) group, 8 mice in each group. After the modeling was successful, it was recorded as the first day. Except for the model group and the commercially available paclitaxel injection group, the rest of the 5 groups were orally administered with the corresponding preparations. The colon cancer mice PTX were administered with a daily dose of 5 mg / kg, continuously administered for 5 days, and then stopped for 5 days, repeated for 1 cycle. The commercially available paclitaxel injection group was injected with paclitaxel injection via the tail vein on the 1st day and the 6th day.

[0132] 4) The small animal live imaging was performed on the 1st, 5th, 10th, 15th and 20th days, and the tumor development in each mouse was dynamically detected. The results are shown in Figure 8 A-F, the average fluorescence intensity of the abdominal part of the mice in the PGS@GLS administration group increased with time, and the increase was the lowest. At the end of the experiment, the average fluorescence intensity was the lowest. On the 21st day, the abdominal tumor of the control group mice significantly increased, and the average fluorescence intensity was 126.7 times that on the 1st day. The abdominal tumor growth of the mice in the paclitaxel intravenous injection administration group, the GLS group, the PGS group and the PGS@GLS group was inhibited to a certain extent, and the average fluorescence intensity was 44.3 times, 37.4 times, 17.0 times and 11.9 times that on the 1st day, respectively. The fluorescence intensity of the mice in the PGS@GLS group was significantly lower than that in the other groups.

[0133] 5) The mice were weighed every other day after administration, and the body weight change was dynamically detected. The results are shown in Figure 9 The body weight of the mice in the PTX intravenous injection group decreased significantly on the 7th day, and showed a downward trend throughout the experiment, indicating that the paclitaxel injection had a large toxic and side effect on the body. The body weight of the mice in the PGS@GLS group was stable, and there was no obvious increase or decrease.

[0134] 6) On the 20th day, the state of the mice was observed, and the mice were comprehensively evaluated according to the body condition score (BCS) in Table 1. The results are shown in Figure 10 The use of PTX alone for intravenous injection can cause the mice to be emaciated and in poor condition. The state of the mice in the PGS@GLS group was better, and the overall score was high. Combined with the body weight change results, it was indicated that the mice in the PGS@GLS group did not appear to be significantly lighter or weak due to tumor burden after administration; Figure 9

[0135] Table 1 Mouse body condition score standard

[0136]

[0137] ​7) On the 21st day, after monitoring the fluorescence intensity by in vivo imaging, the mice were sacrificed, whole blood was collected, and the whole intestine and attached tumors were removed for observation of tumor growth differences between groups. The tumors on the intestine were peeled off and weighed. The results are shown in Figure 11 The average number of intestinal tumors in the PGS@GLS administration group was the least, the volume was smaller, and the average weight of the total tumor was the smallest.

[0138] The mesenteric lymph nodes were peeled off, and tumor samples were selected for digestion to prepare single-cell suspension samples. Fluorescent antibodies were added for incubation, and the proportion of mature dendritic cells (mDCs) and CD8+ T cells in the mesenteric lymph nodes and tumor tissues of the mice in each group was detected by flow cytometry. The results are shown in Figure 12 The proportion of mDCs and CD8+ T cells in the tumor tissues and mesenteric lymph nodes of the mice in the PGS@GLS administration group was significantly increased.

[0139] Tumor samples were selected from each group for fixation, paraffin embedding, and sectioning. Immunofluorescence staining of CRT and HMGB1 was used to analyze the immunogenic cell death fever in the tumor tissues. The results are shown in Figure 13 The CRT fluorescence intensity near the nucleus in the tumor tissues of the mice was significantly enhanced in the PGS@GLS administration group, and the HMGB1 fluorescence intensity on the nucleus was significantly reduced in the PGS@GLS administration group, indicating that PGS@GLS caused CRT everted and HMGB1 released in the nucleus, which could significantly induce immunogenic cell death in tumor cells.

[0140] 8) Tumor samples were selected from each group for fixation, paraffin embedding, and sectioning. Immunofluorescence staining of Ki67 and Tunel was used to analyze tumor cell proliferation and apoptosis. The results are shown in Figure 14 The number of Ki67 positive cells in the tumor tissues of the mice was significantly reduced in the PGS@GLS administration group, which indicated that the tumor cell proliferation rate was relatively low in the PGS@GLS treatment group. In addition, TUNEL staining showed that the tumor cells in the PGS@GLS administration group had the strongest apoptosis signal, which reflected that the promotion of tumor cell apoptosis by PGS@GLS was the most significant.

[0141] 9) Tumor samples were selected from each group for fixation, paraffin embedding, and sectioning. Immunofluorescence staining was used to analyze the infiltration of CD8+ T cells in the tumor tissues. The results are shown in Figure 15 The infiltration of CD8+ T lymphocytes in the mice in the PGS@GLS administration group was the highest.

[0142] 10) The white blood cell (WBC) count, lymphocyte (Lymph) count, red blood cell (RBC) count, hemoglobin (HGB), and platelet (PLT) in the blood of the mice were analyzed to evaluate the safety of the preparation. The results are shown in Figure 16Compared with the control group, the white blood cell, lymphocyte, platelet, red blood cell and hemoglobin count of the PGS@GLS administration group of mice fluctuated within the normal range, showing good biological safety.

[0143] Figure 16 The blood indicators of the PGS@GLS group of mice showed no significant change compared with normal mice. The red blood cell and hemoglobin of the PTX intravenous injection group of mice were significantly lower than those of normal mice.

[0144] 11) The heart, liver, spleen, lung, kidney and tumor tissues of the mice were dissected, fixed, paraffin-embedded and sectioned, and the structure and physiological morphology of each tissue were observed by hematoxylin-eosin staining (H&E staining), and the results are shown in Figure 17 The cell density of the tumor tissue of the PGS@GLS administration group of mice was significantly reduced, and the other main organ tissues were not significantly damaged.

[0145] Figure 18 A schematic diagram of the PGS@GLS composite microparticles prepared in the present application activating cancer immune circulation and inhibiting the development of colorectal cancer.

Claims

1. A Ganoderma lucidum spore composite microparticle containing paclitaxel-loaded Ganoderma lucidum spore oil nanoemulsion, characterized in that: The invention is composed of the following components: a blank carrier of Ganoderma lucidum spores, and a paclitaxel Ganoderma lucidum spore oil self-nanoemulsion; The mass ratio of the blank Ganoderma lucidum spore carrier to the paclitaxel Ganoderma lucidum spore oil nanoemulsion is 1: 5-10; The paclitaxel and ganoderma lucidum spore oil nanoemulsion consists of the following components by mass percentage: paclitaxel 0.08-0.64%, ganoderma lucidum spore oil 10-40%, emulsifier 27-72%, and co-emulsifier 14-60%.

2. The Ganoderma lucidum spore composite microparticles of paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to claim 1, characterized in that: The emulsifier is selected from one of polyoxyethylene 40 hydrogenated castor oil, Tween 80, lauroyl polyoxyethylene 32 glyceride, and caprylic / capric macrogol glyceride.

3. The Ganoderma lucidum spore composite microparticles of paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to claim 1, characterized in that: The co-emulsifier is selected from one of polyethylene glycol 400, propylene glycol monolaurate or Span 80.

4. The method for preparing the Ganoderma lucidum spore composite microparticles from the Ganoderma lucidum spore oil nanoemulsion loaded with paclitaxel according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: mixing Ganoderma lucidum spore oil, an emulsifier, and a co-emulsifier to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate, and adding paclitaxel to the pre-concentrate to obtain a paclitaxel Ganoderma lucidum spore oil self-nanoemulsion; S2: defatting the Ganoderma lucidum spores to obtain defatted Ganoderma lucidum spores; S3: Dispersing the defatted Ganoderma lucidum spores in an alkaline solution and an acid solution, washing and drying, to obtain a Ganoderma lucidum spore blank carrier; S4: Loading the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion onto the Ganoderma lucidum spore blank carrier to obtain Ganoderma lucidum spore composite microparticles loaded with the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion.

5. The preparation method according to claim 4, characterized in that The S2 defatting treatment method is selected from one of solvent extraction, ultrasound-assisted extraction, supercritical CO2 extraction, and gradient centrifugation.

6. The preparation method according to claim 5, wherein The volume-to-mass ratio of the organic solvent to the Ganoderma lucidum spores used in the solvent extraction method is 10-20:1, and the unit is mL / g.

7. The preparation method according to any one of claims 4 to 6, characterized in that The S4 load treatment method is selected from one of a vacuum decompression method, a stirring method and a centrifugal method.

8. Use of the Ganoderma lucidum spore composite microparticles in the paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to any one of claims 1 to 3 in the preparation of pharmaceutical preparations for treating colon cancer or rectal cancer.

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